The steroid hormones (estrogen and progesterone) stimulate growth, maturation and the development of new biochemical capacities in their endocrine target organs. Although it is widely accepted that steroid hormones exert major influences on the transcriptional process, the detailed mechanisms and the associated co-regulator proteins involved in gene transactivation are not yet defined in precise detail. The general objectives of our studies are to define the mechanism of steroid hormones and their receptors in regulating morphologic differentiation and biochemical specialization in target tissues. This will be accomplished by coordinating a network of investigations designed to uncover the mechanisms by which steroid receptors interact with nuclear regulatory proteins (co-activators, co-repressors, general transcription factors, chromatin modifiers, etc.) to effect target gene expression. We will emphasize experimental dissection of the protein-protein interactions which occur inside a living cell and during transcriptional process; we will define the co-regulator's role in modulating transcription and chromatin structure. In addition, we must understand the mechanisms of positive and negative regulation of receptor functional domains effected by various co-regulators. Our studies will utilize the human progesterone receptor, but to establish regulatory concepts, we will carry out selected experiments using human estrogen, thyroid hormone, and retinoic acid receptors. Cell-free binding and transcription approaches, technologies developed over the past five-year period of this grant, will be a corner stone of our methodology; all new concepts will tested finally in the milieu of the intact cell. These studies will involve aspects of nucleic acid and protein biochemistry, protein purification, cell biology and molecular endocrinology. It is expected that the understanding derived from this project will be relevant to the actions of natural sex steroid hormones relative to human physiology. The following proposed studies should also be pertinent to development of more precise theories for the biochemical mechanism of action of intracellular hormones and receptors in general.